Process for producing diaminopimelic acid

ABSTRACT

A PROCESS FOR PRODUCING DIAMINOPIMELIC ACID WHICH COMPRISES CULTURING A MICROOGANISM CAPABLE OF PRODUCING DIAMINOPIMELIC ACID UNDER AEROBIC CONDITIONS IN AN AQUEOUS NUTRIENT MEDIUM CONTAINING AT LEAST ONE HYDROCARBON AS THE MAIN CARBON SOURCE, AND ACCUMULATING AND RECOVERING SAID DIAMINOPIMELIC ACID BROM THE RESULTANT CULTURE LIQUID. HYDROCARBONS WHICH MAY BE EMPLOYED INCLUDE, FOR EXAMPLE, N-PARAFFINS, PREFERABLY HAVING FROM 11 TO 18 CARBON ATOMS KEROSENE, LIGHT OILS, HEAVY OILS, PARAFFIN OILS, NAPHTHA, ETC. PARTICULAR LYSINE-REQUIRING STRAINS OF HYDROCARBON-ASSIMILATING MICROORGANISMS ARE ESPECIALLY PREFERRED IN THE PRECESS, FOR EXAMPLE, ARTHROBACTER PARAFFINEUS ATCC 21087 AND 21088, BREVIBACTERIUM KETOGLUTAMICUM ATCC 21089 AND CORYNEBACTERIUM HYDROCARBOCLASTUS ATCC 21090.

United States Patent 3,719,561 PROCESS FOR PRODUCING DIAMINOPIMELIC ACIDKatsunobu Tanaka, Machida-shi, and Kazuo Oshima and Yoh Tokoro, Tokyo,Japan, assignors to Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan NoDrawing. Continuation-impart of application Ser. No. 663,217, Aug. 25,1967. This application Aug. 7, 1968, Ser. No. 750,780

Claims priority, application Japan, Aug. 26, 1966, ll/55,832 Int. Cl.C12d 13/06 US. Cl. 195-28 R 14 Claims ABSTRACT OF THE DISCLOSURE Aprocess for producing diaminopimelic acid which comprises culturing amicroorganism capable of producing diaminopimelic acid under aerobicconditions in an aqueous nutrient medium containing at least onehydrocarbon as the main carbon source, and accumulating and recoveringsaid diaminopimelic acid from the resultant culture liquor. Hydrocarbonswhich may be employed include, for example, n-paraflins, perferablyhaving from 11 to 18 carbon atoms, kerosene, light oils, heavy oils,paraflin oils, naphtha, etc. Particular lysine-requiring strains ofhydrocarbon-assimilating microorganisms are especially preferred in theprocess, for example, Arthrobacter parafiineus ATCC 21087 and 21088,Brevibacterium ketoglutam icum ATCC 21089 and Carynebacteriumhydrocarboclastus ATCC 21090.

This application is a continuation-in-part of application Ser. No.663,217, filed on Aug. 25, 1967, now abandoned.

This invention relates to a process for producing diaminopimelic acid.More particularly, it relates to a process for the production ofdiaminopimelic acid by fermentation. Even more particularly, theinvention relates to a process for the production of diaminopimelic acidby fermentation with microorganisms in a culture medium containinghydrocarbons as the main source of carbon.

Methods for the production of diaminopimelic acid by the use ofmicroorganisms have been known in the prior art. For example, a methodfor accumulating diaminopimelic acid in a culture medium by the use of alysinerequiring strain of Escherichia (US. Pat. 2,771,396), a methodusing a lysine-requiring mutant strain of Microcaccus glutamicus [AminoAcid, volume 3, p. 83 (1961)] and the like have been reported in theliterature. However, all of these methods involve the production ofdiaminopimelic acid from carbohydrates as the starting material. On theother hand, Yamada et al. have reported the accumulation of variousamino acids from hydrocarbons [Agricultural and Biological Chemistry,vol. 27, pp. 390-395 (1963)]. However, in this latter case, the amountsof amino acids produced are quite small. Accordingly, none of theseprior art methods is satisfactory for the production of diaminopimelicacid on an industrial scale.

Accordingly, one of the objects of the present invention is to providean improved process for the production of diaminopimelic acid whichovercomes the disadvantages and deficiencies of the prior art methods.

Another object of the present invention is to provide a process forproducing diaminopimelic acid by fermentation which may be carried outin an efficacious and simple manner.

A further object of the invention is to provide a process for producingdiaminopimelic acid by fermentation which may be carried outadvantageously on an industrial scale at low cost to give a high yieldof product.

"ice

These and other objects and advantages of the present invention willbecome apparent to those skilled in the art from a consideration of thefollowing specification and claims.

In accordance with the present invention, there has been developed afermentation process for producing diaminopimelic acid, which is animportant precursor of L-lysine, in high yields. This process involvescarrying out fermentation with certain mutant strains, having thecapability of assimilating hydrocarbons, in an aqueous nutrient medium.Hydrocarbons are employed therein as the main source of carbon.

As suitable microorganisms to be employed in the present invention,there may be mentioned, by way of example, the following strains:

Arthrobacter parajfineus No. 2411-U-35 ATCC 21087 Arthrobacterparafizneus No. 2411-U-118 ATCC 21088 Corynebacterium hydrocarboclastusNo. 243 8-U29 ATCC 21090 Brevibacterium ketoglutamicum No. 2473-U-41ATCC 21089 The above strains are capable of assimilating hydrocarbonsand are obtained by the ultraviolet irradiation of the appropriaterespective parent strains. The resultant mutant strains require lysinefor their growth. The parent strains of the mutant microorganisms listedimmediately hereinabove are, respectively, as follows:

Arthrobacter parafjineus No. 2411 ATCC 15591 Arthrobacter parafiineusNo. 2411 ATCC 15591 Corynebacterium hydrocarboclastus No. 2438 ATCCBrevibacterium ketoglutamicum No. 2473 ATCC 15588 As for the culturemedium to be employed in the fermentation, either a synthetic culturemedium or a natural nutrient medium is suitable as long as it containsthe essential nutrients for the growth of the microorganism strainemployed. Such nutrients are well known in the art and includesubstances such as a carbon source, a nitrogen source, inorganiccompounds and the like which are utilized by the microorganism employedin appropriate amounts.

The fermentation in connection With the present invention is conductedin an aqueous nutrient medium containing a hydrocarbon or a mixture ofhydrocarbons as the main carbon source. Such hydrocarbons includestraight and branched-chain parafiins (alkanes) having from 10 to 25carbon atoms, such as n-decane, n-dodecane, n-hexadecane, etc. Otherhydrocarbons which may be employed include cycloparaffins such ascyclohexane and cyclooctane, straightand branched-chain olefins such aspentene-2, hexene-l, octene-l, octene-2, etc., cycloolefins such ascyclohexene, aromatic hydrocarbons such as benzene, o-xylene, etc., andmixtures thereof and crude hydrocarbons such as kerosene, light oils,heavy oils, paraffin oils, gaseous hydrocarbons, etc. Especiallypreferred are n-parafiins having from 11 to 18 carbon atoms, as largequantities of diaminopimelic acid are produced from this startingmaterial.

Small amounts of other carbon sources such as carbohydrates, forexample, glucose, fructose, maltose, sucrose, starch, starchhydrolysate, molasses, etc., or any other suitable carbon source such asglycerol, mannitol, sorbitol, organic acids, etc. may be used in thefermentation medium along with the hydrocarbons. As with thehydrocarbons, these substances may be used either singly or in mixturesof two or more.

As a nitrogen source, various kinds of inorganic or organic salts orcompounds, such as urea and ammonia or ammonium salts such as ammoniumchloride, ammonium sulfate, ammonium nitrate, ammonium phosphate, am-

3 monium carbonate, ammonium acetate, etc., or natural substancescontaining nitrogen, such as cornsteep liquor, yeast extract, meatextract, fish meal, peptone, bouillon, casein hydrolysates, fishsolubles, soy bean meal hydrolysate, etc. may be employed. Thesesubstances may also be used either singly or in combinations of two ormore.

Inorganic compounds which may be added to the culture medium includemagnesium sulfate, sodium phosphate, potassium dihydrogen phosphate,potassium monohydrogen phosphate, iron sulfate or other iron salts,manganese chloride, calcium chloride, etc.

Moreover, it may also be necessary to add certain essential nutrients tothe culture medium, depending upon the particular microorganismemployed, such as amino acids, for example, aspartic acid, threonine,methionine, etc., and/or vitamins, for example, biotin thiamine,cobalamin and the like. In the case of using a purely synthesizedculture medium, thiamine and L-lysine are added thereto.

Fermentation is conducted under aerobic conditions, such as aerobicshaking of the culture or with stirring of a submerged culture, at asuitable temperature, for ex ample, about 25 to 40 C., and a pH of about6 to 8. It is desirable to keep the pH at around neutral (7.0) duringculturing. The pH tends to decrease below 7.0 at the beginning offermentation and it may become necessary to neutralize the culturemedium with a basic substance such as calcium carbonate, ammonia water,sodium hydroxide, ammonium carbonate and the like.

After about 2 to 4 days of culturing under these conditions,considerable amounts of diaminopimelic acid are found to be accumulatedin the fermentation liquor. After the completion of fermentation, thediaminopimelic acid may be separated from the culture liquor byconventional means, such as ion exchange resin treatment, precipitationwith metallic salts, chromatography, or the like. A most advantageousmethod for recovering the diaminopimelic acid involves removing thebacterial cells from the fermentation liquor and recovering the productby an ion exchange resin treatment as described in Example 1. In somecases, small quantities of a-ketoglutaric acid, aspartic acid,L-glutamic acid, L-serine, L-lysine or other amino acids aresimultaneously accumulated in the resultant culture liquor.

The following examples are given merely as illustrative of the presentinvention and are not to be considered as limiting. Unless otherwisenoted, the percentages in the examples and throughout the applicationare by weight per liter of Water.

EXAMPLE 1 Anthrobacter parafiineus No. 2411-U-35 ATCC 21087 (alysine-requiring strain) is employed as the seed bacterium. This strainis cultured in a yeast-bouillon-agar slant at 30 C. for 24 hours inorder to obtain a seed culture.

Twenty ml. portions of a fermentation medium having the followingcomposition are poured into 250 ml. conical flasks, respectively, andsterilized before using:

0.2% KH PO 0.2% Na HPO 0.005% MnSO -4H O 0.001% FeSO -7H O- 0.001%znso.,-7H o 0.5% yeast extract of a mixture of n-parafiins (C -C The pHof the fermentation medium is 7.0. After sterilization, 2% of calciumcarbonate, which has been separately sterilized in the dry state, isadded to the respective flasks.

The seed culture is then inoculated into the fermentation flasks in anamount of 5% by volume. Ammonia water is appropriately added to themedia so as to keep 4 the pH within a range of 6-8 during culturing.Culturing is then carried out with aerobic shaking at 30 C. Table 1shows the results obtained after culturing for 2 and 4 days,respectively.

TABLE 1 Amount of diaminopimelic acid produced, Culturing time, daysmg./m.l.

One liter of the filtrate obtained by removing the bacterial cells fromthe culture liquor which results from culturing at 30 C. for 4 days isadjusted to a pH of 2 and then passed through an ion exchange resin[Diaion SK-l (H-type)]. The resin column is washed with water and elutedwith l N-ammonia water. Fractions showing a positive reaction toninhydrin are collected and concentrated under reduced pressure at 40 C.or less. Decolorization thereof is effected with bone carbon and thenalcohol (methanol or ethanol) is added thereto. As a result, 8.6 gramsof crude crystals of diaminopimelic acid is obtained.

EXAMPLE 2 Brevibacterium ketoglutamicum No. 2473-U-4l ATCC 21089 (alysine-requiring strain) is used as the seed strain. It is culturedunder aerobic conditions with shaking at 30 C. for 24 hours in abouillon medium in order to obtain a seed culture.

Culturing is carried out under the same conditions and in the samemedium as described in Example 1, except that 5% of kerosene instead ofthe n-paraffin mixture is used as the source of carbon. After 4 days ofculturing with aerobic shaking, 2.1 mg./ml. of diaminopimelic acid isfound to be accumulated in the fermentation liquor.

EXAMPLE 3 Culturing is carried out under the same conditions and in thesame medium as described in Example 1, but with Arthrobacterparafi'ineus No. 241 1-U-1l8 ATCC 21088 (a lysine-requiring strain) asthe seed bacterium. After 4 days of culturing, the amount ofdiaminopimelic acid accumulated in the culture liquor is 8.0 mg./ml.

EXAMPLE 4 Culturing is carried out under the same conditions and in thesame medium as described in Example 1, but with Corynebacteriumhydrocarboclastus No. 2438-U29 ATCC 21090 (a lysine-requiring strain) asthe seed bacterium. After 4 days of culturing, the amount ofdiaminopimelic acid accumulated in the culture liquor is 7.5 mg./ ml.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications are intended to be included within the scope of thefollowing claims.

We claim:

1. A process for producing diaminopimelic acid which comprises culturinga microorganism capable of producing diaminopimelic acid and belongingto a species selected from the group consisting of Arthobacterparafiz'neus, Corynebacterium hydrocarboclastus and Brevibacteriumketoglutamicum, said microorganism requiring lysine for its growth underaerobic conditions in an aqueous nutrient medium containing at least onehydrocarbon as the main carbon source, and accumulating and isolatingsaid diaminopimelic acid from the resultant culture liquor.

2. The process of claim 1, wherein said hydrocarbon is an aliphatichydrocarbon of 10 to 25 carbon atoms.

3. The process of claim 1, wherein said hydrocarbon is an n-paraiiin.

4. The process of claim 3, wherein said n-parafi'in contains from 11 to18 carbon atoms.

5. The process of claim 1, wherein said hydrocarbon is selected from thegroup consisting of kerosene, light oils, heavy oils, paraffin oils,naphtha and mixtures thereof.

6. The process of claim 1, wherein said nutrient medium also containsthiamine and L-lysine.

7. The process of claim 1, wherein culturing is carried out at atemperature of from about 25 to 40 C. and at a pH of about 6 to 8.

8. A process for producing diaminopimelic acid which comprises culturinga microorganism selected from the group consisting of Arthrobacterparafiineus ATCC 21087, 'Arthrobacter paraflineus ATCC 21088,Corynebacterium hydrocarboclastus No. 2438-U-29 ATCC 21090, andBrevz'bacterium ketoglutamicum ATCC 21089, under aerobic conditions inan aqueous nutrient medium containing at least one hydrocarbon as themain carbon source, and accumulating and isolating said diaminopimelicacid from the resultant culture liquor.

9. The process of claim 8, wherein culturing is carried out at atemperature of from about 25 to 40 C. and at a pH of about 6 to 8.

10. The process of claim 9, wherein said hydrocarbon is an n-paraffin.

11. The process of claim 10, wherein said n-paraffin contains from 11 to18 carbon atoms.

12. The process of claim 9, wherein said hydrocarbon is selected fromthe group consisting of kerosene, light oils, heavy oils, paraffin oils,naphtha and mixtures thereof.

13. The process of claim 9, wherein said nutrient medium also containsthiamine and L-lysine.

14. The process of claim 9, wherein the isolation of the diaminopimelicacid is carried out by an ion exchange resin treatment.

References Cited UNITED STATES PATENTS 3,222,258 12/1965 Iizuka et a1195--29 OTHER REFERENCES Nakayama et al.: Journal of General AppliedMicrobiology, vol. 7, No. 1, 1961, pp. 41 to 51.

LIONEL M. SHAPIRO, Primary Examiner

